Applications for estrone in preparing anti-ovarian cancer and/or breast cancer products

ABSTRACT

This invention discloses uses for estrone in preparing anti-ovarian cancer and/or breast cancer products. This invention provides uses for estrone in the preparation of products to treat ovarian cancer and/or breast cancer. From carrying out cancer drug repositioning for the FDA- and CFDA-approved drug estrone, experiments for this invention show, based on screening of non-anti-cancer drugs for various cancer cell lines (tissue types) and mutation sites, that estrone has a new use as an anti-ovarian cancer and/or anti-breast cancer medication, thus achieving a new purpose for an old drug.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a national phase entry under 35 U.S.C. § 371 ofInternational Application No. PCT/CN2015/000475, filed Jun. 30, 2015,the content of which is hereby incorporated by reference in itsentirety.

TECHNICAL FIELD

The present invention relates to the technical field of biology, and itparticularly relates to the application of estrone in the preparation ofanti-ovarian cancer and/or breast cancer products.

BACKGROUND

Cancer is the most common as well as the most serious disease thatthreatens human health, and developing effective anti-cancer medicationsis critical to extending patients' lives. Along with the rapiddevelopment of cancer genomics and molecular pharmacology in recentyears, the development of new anti-cancer medications has had relativelygood outcomes. However, since the bottlenecks of large investmentsrequired in the development of new medications and the long-time periodscannot be overcome, as well as the great individual variation in tumorgenetics, many traditional anti-cancer medications are not veryeffective, new medications are expensive, and side effects are not wellunderstood.

In a paper published by the researchers Barabasi A L et. al. in the 2011Nature Reviews Genetics, a molecular network analysis conducted based onGWAS findings and an interactome strategy is expected to reveal new drugtargets and molecular markers for complex diseases, and ultimately toprovide an entirely new understanding of disease pathogenesis andtreatment approaches. Even more noteworthy is that it has beendiscovered in drug repositioning studies that susceptibility geneslocked in by GWAS studies as well as their genes with protein-proteininteraction (PPI) can more easily become indirect targets formedications. This discovery aids in explaining the mechanisms of actionof currently available drugs as well as guiding new drug research. In2014, researchers Okada Y et. al. published a paper in Nature showingthat out of the 101 susceptibility genes for rheumatoid arthritis foundthrough a meta-analysis of GWAS findings, 98 are currently being used asdirect or indirect targets for rheumatoid arthritis medications. Theyalso discovered through drug repositioning research that there aredozens of medications that have been approved for use for otherindications that could be used to treat rheumatoid arthritis.

DISCLOSURE

This research was carried out through integrating cancer gene profilesof the Cancer Gene Census of the Cosmic version 72 cancer histologicaldatabase as well as the protein interactions in the STRING version 10database with Drug Bank Version 4.2, the database of FDA approvedmedications. This obtained candidates for drug repositioning andscreening tests for tumor cell lines were carried out, revealing newanti-cancer drugs. Candidates for tumor suppressing drugs revealed fromthe cancer cell line screening are as follows:

nicardipine, promethazine, estrone, sulindac, etonogestrel,levonorgestrel, mesalazine, indomethacin, sulfasalazine, palsalazide,irbesartan, ibuprofen, isoprenaline, and pentosan polysulfate.

The primary goal of this invention is to provide a new use for estrone.

This invention provides uses for estrone in the preparation of productsto treat ovarian cancer and/or breast cancer.

The second goal of this invention is to provide a new use for estrone.

This invention provides uses for estrone in the preparation of productsto inhibit the proliferation of ovarian cancer cells and/or breastcancer cells.

The third goal of this invention is to provide a new use for estrone.

This invention provides uses for estrone in the preparation of productsto reduce IC50 values in ovarian cancer cells and/or breast cancercells.

Uses of estrone in treating ovarian cancer and/or breast cancer are alsowithin scope of protection of this invention.

Uses of estrone in inhibiting the proliferation of ovarian cancer cellsand/or breast cancer cells are also within scope of protection of thisinvention.

Uses of estrone as a medication for treating ovarian cancer and/orbreast cancer are also within scope of protection of this invention;

Uses of estrone as a medication for inhibiting the proliferation ofovarian cancer cells and/or breast cancer cells are also within scope ofprotection of this invention.

In the application above, the ovarian cancer cells mentioned are SKOV-3;the breast cancer cells mentioned are HCC1395.

In the uses above, the product is a medication or reagent kit.

The fourth goal of this invention is to provide a kind of product.

The active ingredient in the product of this invention is Estrone, andit has at least one of the following functions:

1) Treatment of ovarian cancer and/or breast cancer;

2) Inhibition of the proliferation of ovarian cancer cells and/or breastcancer cells;

3) Reduction of IC50 values of ovarian cancer cells and/or breast cancercells;

In the products described above, the ovarian cancer cells mentioned areSKOV-3; the breast cancer cells mentioned are HCC1395.

In the product above, the product is a medication or reagent kit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a distributed 96-well drug screening culture plate.

FIG. 2 is estrone sensitivity to ovarian cancer; EC50=16.4284;IC50=21.5327; R²=0.9875.

FIG. 3 is estrone sensitivity to breast cancer; EC50=74037.5043;IC50=67.9510; R²=0.9909.

BEST MODE TO CARRY OUT THE INVENTION

Unless otherwise specified, the experimental methods employed in thefollowing examples are standard methods.

Materials, reagents, etc. used in the following examples are allcommercially available unless otherwise specified, and the experimentalmethods employed in the following examples are standard methods.

The analyte drug in the following examples is Estrone, and its chemicalcomposition is as follows:

It is a drug bank product with a catalog number of DB00655.

In the examples below, the product sources for the SKOV-3 ovarian cancercells, the NCI-H1437 human lung cancer cells, the NCI-H460 large-celllung cancer cells, and the HCC1395 breast cancer cells are as follows:

SKOV-3 ATCC HTB-77 NCI-H1437 ATCC CRL-5872 NCI-H460 ATCC HTB-177 HCC1395ATCC CRL-2324

The primary instruments and materials in the examples below are:

DMSO (from Sigma, Cat. No. D4540)

96-well clear bottom cell culture plates (from Corning, Cat. No. 3610)

CellTiter Glo reagent kit (from Promega, Cat. No. G7573)

Doxorubicin positive medication (from MCE, Cat. No. HY-15142)

Fetal Bovine Serum (from Gibco, Cat#10099141)

100 mm petri dish (from Corning, Cat#430167)

RPMI-1640 medium (from Gibco, Cat# A1049101)

DMEM medium (from Gibco, Cat#11995081)

DMEM/F12 medium (from Gibco, Cat#11330057)

EMEM medium (from Gibco, Cat#10370021)

Multidrop 384 cell dispensers (Thermo, Cat#5840150)

EnSpire multi-function plate reader (Perkin Elmer, Cat#2300-001M)

In example 1, CELLTITER-GLO was used to test estrone against ovariancancer and/or breast cancer

A. Test Plate Preparation

1. Cell Plating

a) The complete medium required for each cell was prepared.

b) Before beginning the experiment, the name of the drug screened forthe cells marked on the 100 mm petri dish was confirmed as well asinformation such as the passage time and number of passages to ensurethe experiment was error-free.

c) Refer to steps d) through i) for procedures for adherent cells; referto steps j) through l) for procedures for suspension cells.

d) When using aseptic technique, a vacuum pump was used to draw the cellculture medium.

e) 2 ml of a sterile PBS solution was used to rinse the cell surface,and a vacuum pump was used to aspirate the PBS waste.

f) A 1 ml 0.25% (w/v) Trypsin-0.038% (w/v) EDTA solution was gentlyadded to the petri dish for cell digestion, and after gently mixingseveral times, the solution covered the cell surfaces. The status ofcell digestion was observed under an inverted microscope, and thetrypsin digestion effect was terminated when cell shedding was about tooccur.

g) 5 ml of pre-warmed 37° C. complete medium was added to the petridishes, and a pipette was used to gently dissociate the cells in orderfor them to shed from the bottom of the petri dish.

h) The cells were suspended and transferred to a 15 ml or 50 ml sterilecentrifuge tube and they were centrifuged at 1000 rpm for 5 minutes.

i) A vacuum pump was used to aspirate the medium with aseptic technique.5 ml of pre-warmed 37° C. complete medium was used to resuspend the cellsediment, and it was gently dissociated to mix evenly.

j) A pipette was used to gently dissociate the cells so that they fullyshed from the bottom of the petri dish.

k) The cells were suspended and transferred to a 15 ml or 50 ml sterilecentrifuge tube and they were centrifuged at 1000 rpm for 5 minutes.

l) A vacuum pump was used to aspirate the medium with aseptic technique.5 ml of pre-warmed 37° C. complete medium was used to resuspend the cellsediment, and it was gently dissociated to mix evenly.

m) A cell counter was used to count the number of suspended cells andadjust the cell suspension to an appropriate density in the plate tocarry out cell plating experiments.

SKOV-3 cells were handled as described above, and a SKOV-3 96-well cellculture plate was obtained.

The complete medium for SKOV-3 cells was McCOY'S 5A (a live product),Cat#16600082, with a cells/well density of 12000.

2. The drug analyte estrone was prepared and administered (200× finalconcentration):

1) The master plate for the drug analyte estrone was prepared

a) DMSO was used to dilute the analyte estrone to 20 mM for use.

b) 79 μL of the 20 mM analyte prepared in step a) was added to the firstwell in the first row of the dilution plate, and then 54 μL of DMSOsolution was added to the second through ninth wells of the first row.25 μL of solution was aspirated from the first to the second well, andafter mixing well 25 μL of solution was aspirated from the second to thethird well, and this was repeated until the 9th well in order to ensurethat 3.16 dilution of the medication would be carried out one-by-one.

2) Doxorubicin positive medication (MCE, Cat. No. HY-15142) master platepreparation

a) DMSO was used to dilute Doxorubicin positive medication to 6 mM foruse.

b) The 6 mM Doxorubicin positive medication solution was added to thedilution plate, and the DMSO solution was incrementally added to theanalyte medication to 1:3.16.

3. Drug Working Board Preparation and Dosing

a) The analyte drug and the positive drug sampling template is as shownin FIG. 1, in which S1208: Positive medication Doxorubicin, DMSO:Positive control well, Cpd 1, 2, 3: Analyte drug, DMSO finalconcentration of 0.5% (DMSO compatibility).

b) 95 μl of cell-specific complete medium was added to the workingplate, each medication to 9 wells. A multi-channel pipettor was used totransfer a series of 5 μl (9 wells) of the diluted solution of theanalyte drug and positive medication Doxorubicin (10× finalconcentration) from the working plate, achieving cell culture media ofvarying concentrations.

c) The SKOV-3 96-well cell plates prepared in step 1 were removed fromthe incubator, and 10 μl of the cell culture media (10× finalconcentration) with varying drug concentrations as described in b) abovewas added to the SKOV-3 96-well cell culture plate row-by-row as shownin FIG. 1. It was placed into a CO₂ incubator at 37° C. for 72 hours,obtaining the SKOV-3 96-well drug screening plate.

Wells with no medication added acted as controls.

The final concentration and dosing of the analyte drug, positivemedication Doxorubicin, and control in the 96-well plates were asfollows:

The final concentrations (μM) of the analyte drug in wells 2-10 in FIG.1 are, in order: 100, 31.64557, 10.01442, 3.16912, 1.002886, 0.317369,0.100433, 0.031783, 0.010058;

The final concentration (μM) of the positive medication Doxorubicin inwells 2-10 in FIG. 1 are, in order: 30, 9.493671, 3.004326, 0.950736,0.300866, 0.095211, 0.03013, 0.009535, 0.003017;

In addition, the S1208 well in the 96-well plate (E1-H1 and A12-D12): 10μl of the final concentration 100 μM Doxorubicin solution (solventcontaining 0.5% DMSO complete culture medium solution), DMSO wells(A1-D1, E12-H12, and A11-H11): 10 μl containing 0.5% DMSO completeculture medium solution.

B. CELLTITER-GLO Luminescent Cell Viability Assay System

1. CellTiter-Glo Reagent Preparation

a) The CellTiter-Glo reagent buffer was thawed before using andstabilized to room temperature for use.

b) The CellTiter-Glo reagent frozen substrate was thawed before usingand stabilized to room temperature for use.

c) 100 ml of stabilized CellTiter-Glo buffer was added to the containerwith CellTiter-Glo reagent frozen substrate to adequately resuspend itto form an enzyme/substrate mixture, also referred to as theCellTiter-Glo assay reagent.

d) It was gently mixed and vortexed and inverted multiple times toachieve a uniform solution. In general, the CellTiter-Glo substratereagent will adequately dissolve within 1 minute. It is storedseparately in low-light conditions at −20° C. to await use, and freezingrepeatedly should be avoided.

2. Testing

a) Before testing, the SKOV-3 96-well drug screening plate described in3 above was stabilized to room temperature for 20-30 minutes.

b) An inverted microscope was used to observe the conditions of eachgroup of cells in the culture plate and their death patterns, and anyabnormal conditions were noted and retested.

c) 100 μl of CellTiter-Glo reagent (prepared as described in 1 above)was added to all drug screening plates and mixed evenly.

d) It was thoroughly oscillated in a 96-well microplate oscillator for 2minutes to allow the cells to undergo full lysis.

e) It was stored away from light at room temperature for 15 minutesbefore carrying out luminescent signal detection to ensure signalstability.

f) An EnSpire multi-function plate reader was used at 570 nm to read theluminescent signals.

g) Data was processed and analyzed.

The results of the SKOV-3 96-well drug screening plate are shown in FIG.2.

The IC50 value was calculated; results are shown in Table 1.

The same method was used to test Estrone's action on the IC50 value ofHCC1395 breast cancer cells; results are shown in Table 1 and FIG. 3.

Breast cancer cells HCC1395, complete medium basis of RPMI-1640, (a liveproduct), Cat# A1049101, with a cells/well density of 6000.

The same method was used to test Estrone's action on the IC50 value ofNCI-H460 human lung cancer cells and NCI-H460 large-cell lung cancercells; results are shown in Table 1.

It is evident that estrone has a specific inhibitory effect on theproliferation of ovarian cancer cells and it can be used as a medicationfor ovarian cancer treatment.

TABLE 1 IC50 values of various cells under the effect of Estrone CellsIC50 value SKOV-3 21.5327 NCI-H460 100 NCI-H1437 100 HCC1395 67.9510

INDUSTRIAL APPLICATIONS

From carrying out cancer drug repositioning for the FDA- andCFDA-approved drug Estrone, experiments for this invention show, basedon screening of non-anti-cancer drugs for various cancer cell lines(tissue types) and mutation sites, that estrone has a new use as ananti-ovarian cancer and/or anti-breast cancer medication, thus achievinga new purpose for an old drug.

What is claimed is:
 1. A method for treating ER-negative breast cancerin a subject comprising administering an effective amount of estrone toa subject in need thereof.
 2. The method of claim 1, wherein theER-negative breast cancer is a primary ductal carcinoma.
 3. A method ofinhibiting the proliferation of ER-negative breast cancer cellscomprising contacting the cells with an effective amount of estrone. 4.The method of claim 3, wherein the breast cancer cells are HCC1395cells.